This invention is generally in the field of tryptophan transporters, and more specifically is drawn to a novel tryptophan transport system.
Tryptophan occupies a unique position at the interface between the immune system and cellular metabolism. It is the only amino acid whose level is specifically and selectively modified in response to signals of infection and inflammation. This is accomplished by the enzyme indoleamine 2,3-dioxygenase (IDO), which rapidly and selectively degrades tryptophan following induction by interferon-xcex3 (IFNxcex3) and other proinflammatory signals (Taylor and Feng, 1991 FASEB J. 5, 2516-2522). The IDO system, whose origins extend back to invertebrates (Suzuki, et al., 1996 Biochem. Biophys. Acta 1308, 41-48; Suzuki, 1994 J. Prot. Chem. 14, 9-13) is hypothesized to inhibit the replication of intracellular pathogens (Gupta, et al., 1994 Infect. Immun. 62, 2277-2284; Thomas, et al., 1993 J. Immunol. 150, 5529-5534) and to participate in the antiproliferative effect of interferons on host cells (Aune and Pogue, 1989 J. Clin. Invest. 84, 863-875; Feng and Taylor 1989, Proc. Natl. Acad. Sci. USA, 86, 7144-7148; Ozaki, et al., 1988, Proc. Natl. Acad Sci. USA 85, 1242-1246). Tryptophan is also unique in that it is the only amino acid whose entry into the protein synthetic pathway is regulated by the immune system. Like IDO, the gene for tryptophanyl-tRNA synthetase is highly induced by IFNxcex3 (Fleckner, et al., 1991 Proc. Natl. Acad. Sci. USA 88, 11520-11524; Fleckner, et al., 1995 Cytokine 7, 70-77; Rubin, et al., 1991 J. Biol. Chem. 266, 24245-24248), which has been proposed to assist cells in competing for scarce supplies of tryptophan (Kisselev, 1993 Biochimie 75, 1027-1039). These observations indicate that tryptophan metabolism plays an important role in host defense.
Activated macrophages display a marked induction of the tryptophan catabolic pathway (Werner, et al., 1987 Life Sci. 41, 273-280; Carlin, et al., 1989, J. Leuk. Biol. 45, 29-34). These cells are capable of reducing the tryptophan concentration in their local microenvironment to such a low level that cellular proliferation becomes impossible. This mechanism underlies the ability of certain types of macrophages (Munn, et al., 1996 J. Immunol. 156, 523-532) to inhibit T cell activation. In order to achieve this effect, however, macrophages must reduce the tryptophan concentration to the low nanomolar range (less than 50 nM), three orders of magnitude below its normal level. Macrophages can continue to take up tryptophan efficiently at very low substrate concentrations, even when tryptophan is competing with multiple other amino acids, present at much higher concentrations, for transport into the cell.
Based on this analysis, it is postulated that there must be a specific, high affinity tryptophan transporter.
It is therefore an object of the present invention to provide a high-affinity amino acid transport system that is highly specific for tryptophan.
It is another object of the present description to provide methods and reagents for use in isolating and characterizing the cDNA and amino acid sequence of high affinity tryptophan transporters.
It is yet a still further object of the present invention to provide methods and reagents for designing and isolating molecules and drugs that can stimulate or inhibit the binding or transport of tryptophan with high affinity tryptophan transporters.
A high affinity and extremely selective tryptophan transport system present in human monocyte-derived macrophages is disclosed. Human monocyte-derived macrophages include two distinct transporters, a high affinity (Km=290xc2x1160 nM) transporter that is highly specific for tryptophan and a low affinity (Km=27xc2x14 xcexcM) transporter that is less specific for tryptophan, consistent with the known system L. The tryptophan transport system is predominantly (86%) sodium-independent. The high-affinity system is very specific for tryptophan and shows no transport of any other essential amino acids in the tryptophan transport concentration range. This high-affinity system is expressed at very low levels in fresh monocytes, but undergoes a 10-30 fold induction during macrophage differentiation. This high affinity, tryptophan-selective transport system allows macrophages to take up tryptophan efficiently under conditions of very low substrate concentration, such as can occur at sites of inflammation.
Methods for the isolation of the cDNA that codes for this high affinity tryptophan transport system are described along with methods for the purification of the high affinity transporter. Methods for obtaining and using molecules which interact with the high affinity transporter are also described. Molecules which are obtained by their competitive binding properties with tryptophan for the high affinity transporter tryptophan binding site are also described. Also described are compositions, and methods for using these compositions, which do not competitively interact with tryptophan for the high affinity transporter tryptophan binding site.